Variable epitope library compositions and methods of therapeutic and prophylactic use

ABSTRACT

The present disclosure relates to compositions and methods for targeting antigenically variable pathogens and diseases. Embodiments of the present disclosure involve of the construction of variable epitope libraries (VELs) containing mutated versions of epitopes derived from antigens associated with various diseases for treating subjects in both therapeutic and prophylactic settings. The present disclosure also provides compositions and methods for the production of VELs based on CTL-derived epitopes of survivin, an oncogenic inhibitor-of-apoptosis. Given the large number of potential epitopes expressed in tumors, and the dynamic nature of the tumor epitope landscape, there is a need to develop compositions and methods for targeting various antigenic epitopes to counteract immune escape.

CROSS-REFERENCE TO RELATED APPLICATIONS

The instant application is a continuation of U.S. application Ser. No.14/991,807, filed Jan. 8, 2016, which claims the benefit of U.S.Provisional Patent Application No. 62/101,874, filed Jan. 9, 2015. Theseapplications are incorporated herein by reference in their entirety forall purposes.

FIELD OF THE INVENTION

Embodiments of the present disclosure relate to compositions and methodsfor targeting antigenically variable pathogens and diseases. Certainembodiments disclosed herein relate to construction of variable epitopelibraries (VELs) containing mutated versions of epitopes derived fromantigens associated with various diseases of use for treating subjectsin both therapeutic and prophylactic settings. Other embodiments providecompositions and methods for the production of VELs based on CTL-derivedepitopes of survivin, an oncogenic inhibitor of apoptosis.

BACKGROUND OF THE INVENTION

The immune system protects a subject from possibly harmful substances byrecognizing and responding to antigens. Antigens are substances,typically peptides, on the surface of cells, viruses, fungi, orbacteria. Nonliving substances such as toxins, chemicals, drugs, andforeign particles can also be antigens. The immune system recognizes anddestroys substances that contain antigens. An epitope, also referred toas an antigenic determinant, is a portion of an antigen that isrecognized by various molecules and cells that make up a subject'simmune system (e.g., antibodies, T cells, B cells). An epitope is aspecific peptide region of the antigen to which an antibody binds.T-cell epitopes, for example, are presented on the surface ofantigen-presenting cells where they are typically bound to MHC molecules(major histocompatibility complex). An immune response is initiatedfollowing T cell recognition of antigen peptides in the context of selfMHC molecules, and generally takes place in one of the host's secondarylymphoid compartments. Cellular activation is triggered by the bindingof antigen to the T cell receptor (TCR), forming an antigen/TCR complexwhich transduces the antigen-specific extracellular stimulation acrossthe plasma membrane, and generates intracellular signals which includethe activation of protein kinase C and the increases in intracellularcalcium. While signal transduction can lead to T cell unresponsiveness,positive signal transduction events trigger a series of additionalbiochemical processes that lead to an immune response in a subject.

One obstacle in the advancement for developing vaccines againstpathogens with genetic variability is immune escape. Typically, immuneescape involves amino acid substitutions in specific epitopes of apathogenic antigen recognized by the host immune system (e.g., CTL, Thand B epitopes). Despite the degenerate nature of the interactionsbetween a TCR of T cells and MHC/peptide complex on antigen-presentingcells, the majority of circulating variants are not recognized by CTLs.This may explain the immune system's failure in clearing or containingvarious pathogens. The ability of pathogens to escape immunity bymutating amino acids in epitopes or flanking regions (affecting thecorrect epitope processing) is an ongoing and dynamic process involvingcomplex viral-host interactions. Other factors affecting the immuneescape phenomenon include viral fitness, cost of mutations, immunepressure exerted by the host, host genetic factors, and viral load.

In the field of cancer epitope vaccines, the modified, optimized orvariant peptides, also known as altered peptide ligands (APLs),mimotopes, heteroclitic peptides or peptide analogues, bearing mutatedversions of natural epitopes derived from tumor-associated antigens(TAAs) are considered to be promising candidates for the development ofvaccines. Comprehensive screening strategies, such as testing virtuallyevery single amino acid substitution within an epitope by geneticscreen, may lead to identification of superagonist APLs capable ofeliciting potent anti-tumor patient-specific CTL responses when thenative or wild type (WT) tumor-associated epitope fails. CentralTCR-contact residues of antigenic peptides can be replaced even bynon-peptidic units without loss of binding affinity to majorhistocompatibility complex (MHC) class-I molecules and T-cell triggeringcapacity. However, there are a very large number of potential epitopesexpressed in tumors that are encoded by non-primary open reading frame(ORF) sequences (frame-shift mutations) or derived from othernon-traditional sources, such as transcriptional/translationalmechanisms or splicing events, collectively referred as epitopes derivedfrom defective ribosomal products (DRiPs).

SUMMARY

Embodiments disclosed herein provide variable epitope library (VEL)vaccine compositions composed of one or more isolated peptides havingamino acid sequences corresponding to an epitope of a pathogenicantigen, the one or more peptides spanning from about 7 to about 50total amino acids, wherein from about 1% to about 50% of the total aminoacids of the one or more peptides are variable amino acids, and whereinthe composition generates an immune response when administered to asubject. Embodiments of the VEL vaccine composition also include apharmaceutically acceptable excipient and/or adjuvant.

Embodiments disclosed herein also provide VELs comprising one or moresynthetic isolated peptides having amino acid sequences corresponding toan epitope of a pathogenic antigen, the one or more peptides spanningfrom about 7 to about 50 total amino acids, wherein from about 1% toabout 50% of the total amino acids of the one or more peptides arevariable amino acids.

Other embodiments disclosed herein can provide for a VEL vaccinecomposition comprising one or more isolated polynucleotides encoding oneor more peptides having amino acid sequences corresponding to an epitopeof a pathogenic antigen, the one or more polynucleotides encoding one ormore peptides spanning from about 7 to about 50 total amino acids,wherein the one or more polynucleotides have mutations that encodevariable amino acids in from about 1% to about 50% of the total aminoacids of the one or more peptides, and wherein the composition generatesan immune response when administered to a subject. In accordance withthese embodiments, the VEL vaccine compositions can further include apharmaceutically acceptable excipient and/or adjuvant.

Embodiments disclosed herein can include a VEL comprising one or moreisolated polynucleotides encoding one or more peptides having amino acidsequences corresponding to an epitope of a pathogenic antigen, the oneor more polynucleotides encoding one or more peptides having from about7 to about 50 total amino acids, further the one or more polynucleotidescan contain one or more mutations that encode variable amino acids ofabout 1% to about 50% of the total amino acids of the one or morepeptides.

VEL libraries and VEL vaccine compositions disclosed herein can includeepitopes derived from pathogenic antigens of a survivin-derived CTLepitope. The VEL libraries and vaccine compositions can include variableamino acids that can be any of the 20 naturally occurring amino acids orderivatives thereof. The variable amino acids in the VEL libraries andvaccine compositions can be from about 10% to about 50% of the totalamino acids of the one or more peptides, such that the complexity of thelibrary or vaccine composition can be about 208 synthetic peptides.

Embodiments of the present disclosure provide for compositions andmethods of producing VELs based on CTL-derived epitopes of survivin, anoncogenic inhibitor of apoptosis. VELs containing CTL-derived epitopesof survivin can be based for example, on the survivin-derivedH-2Dd-restricted wild-type CTL epitope, GWEPDDNPI (SEQ ID NO: 2). Insome embodiments, VELs containing CTL-derived epitopes of survivin canbe based, for example, on the epitope GWXPXDXPI (SEQ ID NO: 1), where Xis any one of the 20 naturally occurring amino acids or derivativesthereof.

VEL libraries and VEL vaccine compositions disclosed herein can beadministered to a subject prophylactically or therapeutically to treat,prevent, and/or reduce the risk of developing various diseases fromvarious pathogens, such as a cancerous tumor. Methods disclosed hereincan include methods of treating cancer in a subject including injectinga variable epitope library vaccine composition having one or moreisolated peptides with amino acid sequences corresponding to a survivinCTL epitope, the one or more peptides having from about 7 to about 50total amino acids, wherein from about 1% to about 50% of the total aminoacids of the one or more peptides are variable amino acids, and apharmaceutically acceptable excipient and/or adjuvant. In accordancewith these embodiments, when introduced to a subject, these compositionscan generate an immune response. Methods disclosed herein includetreating a subject diagnosed with cancer with one or more VELcompositions, where the cancer includes one or more tumors and thecomposition administered to the subject reduces the mass or volume ofthe one or more tumors.

In some embodiments, VEL libraries and VEL vaccine compositionsdisclosed herein can be produced by obtaining the amino acid sequence ofone or more peptides corresponding to a survivin CTL epitope, andsynthesizing the one or more peptides corresponding to a survivin CTLepitope, wherein the one or more peptides comprise from about 7 to about50 total amino acids, and wherein from about 1% to about 50% of thetotal amino acids of the one or more peptides are variable amino acids.Embodiments can include combining the one or more peptides correspondingto a survivin CTL epitope into a mixture and adding at least onepharmaceutically acceptable excipient, agent and/or adjuvant to themixture of one or more peptides corresponding to a survivin CTL epitope.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe subject matter of the disclosure, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting. Other features andadvantages of the disclosure will be apparent from the followingdetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the instant specification and areincluded to further demonstrate certain aspects of particularembodiments herein. The embodiments may be better understood byreference to one or more of these drawings in combination with thedetailed description presented herein. This patent or application filecontains at least one drawing executed in color. Copies of this patentor patent application publication with color drawing(s) will be providedby the Office upon request and payment of the necessary fee.

FIGS. 1A-1D are schematic diagrams illustrating the relative timing oftumor challenge, vaccination (prime and booster), and tumor isolationand analysis in experiments testing the efficacy of the phage displayand synthetic peptide variable epitope libraries (VELs), according toone embodiment of the present disclosure.

FIG. 2A is a graphical representation of the efficacy of prophylacticadministration of a phage display VEL based on a survivin-derived CTLepitope for reducing 4T1-induced tumor growth, according to oneembodiment of the present disclosure.

FIG. 2B is a graphical representation of the efficacy of therapeuticadministration (single injection) of a phage display VEL based on asurvivin-derived CTL epitope for reducing 4T1-induced tumor growth,according to embodiments herein.

FIG. 2C is a graphical representation of efficacy of therapeuticadministration (multiple injections) of a phage display VEL based on asurvivin-derived CTL epitope for reducing 4T1-induced tumor growth,according to embodiments herein.

FIG. 2D is a graphical representation of efficacy of therapeuticadministration (single injection) of a synthetic peptide VEL based on asurvivin-derived CTL epitope for reducing 4T1-induced tumor growth,according to embodiments herein.

FIG. 2E is a graphical representation of efficacy of therapeuticadministration (multiple injections) of a synthetic peptide VEL based ona survivin-derived CTL epitope for reducing 4T1-induced tumor growth,according to embodiments herein.

FIG. 3A is a graphical representation of the efficacy of a phage displayVEL based on a survivin-derived CTL epitope to induce a cellular immuneresponse in immunized mice (FSVL or FSWT) after 4T1-induced tumorchallenge, according to embodiments herein.

FIG. 3B is a graphical representation of the efficacy of prophylacticadministration (single injection) of a phage display VEL based on asurvivin-derived CTL epitope for inducing a cellular immune responseafter 4T1-induced tumor challenge, according to embodiments herein.

FIG. 3C is a graphical representation of the efficacy of therapeuticadministration (single injection) of a phage display VEL based on asurvivin-derived CTL epitope for inducing a cellular immune response,according to embodiments herein.

FIG. 3D is a graphical representation of the efficacy of a phage displayVEL based on a survivin-derived CTL epitope to induce a cellular immuneresponse in non-immunized mice after 4T1-induced tumor challenge,according to embodiments herein.

FIG. 4A is a graphical representation of the efficacy of therapeuticadministration of specific phage display VEL epitopes to induceactivation of subpopulations of T-cells after 6 hours of stimulationwith phage clones, according to embodiments herein.

FIG. 4B is a graphical representation of the efficacy of therapeuticadministration of specific phage display VEL epitopes to induceactivation of subpopulations of T-cells after 72 hours of incubationwith phage clones, according to embodiments herein.

FIG. 4C is a graphical representation of the efficacy of prophylacticadministration of specific phage display VEL epitopes to induceactivation of subpopulations of T-cells after 72 hours of incubationwith phage clones, according to embodiments herein.

FIG. 4D is a graphical representation of the efficacy of prophylacticadministration of specific phage display VEL epitopes to induceactivation of subpopulations of T-cells after 72 hours of incubationwith phage clones, according to embodiments herein.

While the disclosure is amenable to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and are described in detail herein.

DETAILED DESCRIPTION

Embodiments of the present disclosure relate to compositions and methodsfor targeting antigenically variable pathogens and diseases. Certainembodiments disclosed herein relate to construction of variable epitopelibraries (VELs) containing mutated versions of epitopes derived fromantigens associated with various diseases of use for treating subjectsin both therapeutic and prophylactic settings. Other embodiments providecompositions and methods for the production of VELs based on CTL-derivedepitopes of survivin, an oncogenic inhibitor of apoptosis. Given this,and the dynamic and elusive nature of the tumor epitope landscape, thereis a need to develop compositions and methods for targeting variousantigenic epitopes to counteract immune escape and provide alternativetreatments to these conditions.

Embodiments of the present disclosure provide for VEL compositions andmethods of use for treatment of disease. In certain embodiments, acomposition may include a synthetic peptide. In accordance with theseembodiments, the synthetic peptide may include at least one epitope of apathogen- or disease-specific polypeptide, where at least one amino acidresidue of the peptide is substituted with each of the other nineteencommon amino acid residues. In another embodiment, the presentdisclosure provides for VEL compositions that can include nucleic acidsequences or nucleic acid sequence derivatives. In accordance with thisembodiment, the nucleic acid sequences or nucleic acid sequencederivatives may encode a peptide having at least one epitope of apathogen- or disease-specific polypeptide, where at least one amino acidresidue of the encoded peptide is substituted with each of the othernineteen common amino acid residues.

In one example, VEL compositions disclosed herein may be prepared byexpression in a bacterial, viral, phage display, or eukaryoticexpression system. In another example, the VEL compositions may beexpressed and displayed on the surface of a recombinant bacteriophage,bacterium or yeast cell. In accordance with these embodiments, thecomposition of an epitope of a pathogen-specific nucleic acid orpolypeptide disclosed herein may be selected from one or more epitopesof Human Immunodeficiency Virus (HIV), Simian Immunodeficiency Virus(SIV), Hepatitis A, Hepatitis B, Hepatitis C, rhinovirus, influenzavirus, Plasmodium falciparum, tuberculosis, cancer (e.g., breastcancer), and infections salmon anemia virus (ISAV). Alternatively, theepitope of a disease-specific polypeptide may be one or more epitopes ofa tumor associated antigen (TAA).

In another embodiment, a method for preparing and using a variableepitope library may include preparing the variable epitope library(VEL), injecting the library into a subject and inducing an immuneresponse in the subject against the VEL. In accordance with thisembodiment, preparing a VEL may include preparing a VEL bearing epitopesof a pathogen-specific polypeptide. In another embodiment, the methodmay include preparing a VEL where the VEL bears epitopes of adisease-specific polypeptide. In one example, inducing an immuneresponse in a subject may include inducing an immune response effectiveto protect a subject against infection with a pathogen. In anotherexample, inducing the immune response may include inducing the immuneresponse effective to treat a subject infected with a pathogen or toprotect the subject against onset of a condition such as cancer.

Variable Epitope Libraries (VELs)

Genetic variability of many pathogens and disease-related antigens canresult in the selection of mutated epitope variants able to escapecontrol by immune responses. This can be a major obstacle to vaccinedevelopment against certain pathogens. Embodiments herein relate toimmunogens composed of variable epitope libraries derived from pathogensand disease-related antigens with genetic/antigenic variability in orderto advance strategies for overcoming these issues with pathogenicorganisms.

An immunogen vaccine composition that includes variable disease epitopesis referred to as a variable epitope library (VEL). VELs can be about 7to about 50 amino acids (aa) or amino acid residues in length. Forexample, the polypeptides including a VEL can be P₁P₂P₃ . . . P_(n),where the numbers represent positions (P) of the various wild type aminoacids, and where “n” represents the total polypeptide length and theposition of the last amino acid. In various embodiments disclosedherein, at least one amino acid and as many as 90% of wild type aminoacid residues can be randomly replaced by any of the 20 naturallyoccurring amino acid residues. As one of skill in the art would readily,VELs and VEL compositions are neither natural products nor naturallyoccurring, and VELs and VEL compositions are made-up of polypeptidesthat are neither natural products nor naturally occurring. Also, as oneof skill in the art would readily recognize based, VELs and VELcompositions include polypeptides that are not yet known or identified,which enables VELs and VEL compositions to induce a broad range ofprotective immune responses when introduced to a subject before one ormore mutated epitopes (before infection) emerges or when the amount ofone or more mutated epitopes is low (early stages of infection and/ordisease progression).

In alternative embodiments, VELs can contain nucleic acid sequencemolecules comprising from about 20 to about 200 individual nucleotidesthat encode the variable epitope polypeptides. In other embodiments,VELs can contain one or more polypeptide molecules where from about 10%to about 50% of the total amino acids of the one or more polypeptidemolecules are variable amino acids (replaced by any of the 20 naturallyoccurring amino acid residues or a derivative of a naturally occurringamino acid). In other embodiments, VELs can contain one or morepolypeptides in which from about 20% to about 50% of the total aminoacids of the one or more peptides are variable amino acids. In certainembodiments, VELs can contain one or more polypeptides in which fromabout 30% to about 50% of the total amino acids of the one or morepeptides are variable amino acids. In yet other embodiments, VELs cancontain one or more polypeptides in which from about 20% to about 40% ofthe total amino acids of the one or more peptides are variable aminoacids.

For example, VELs and VEL vaccine compositions disclosed herein can becomposed of a decapeptide, P₁P₂P₃P₄P₅P₆P₇P₈P₉P₁₀, that can berepresented as P₁X₂P₃X₄P₅X₆P₇X₈P₉X₁₀ where X can be any of the 20naturally occurring amino acids or derivatives of a naturally occurringamino acid, and where P can be an amino acid that is the same amino acidas that of the wild type epitope at that position. Similarly, anotherversion of VEL based on the same decapeptide may be constructed byreplacing wild type amino acid residues by X residues at odd positionsand leaving this time wild type residues at even positions. While inthese two particular decapeptide-based VELs each individual librarymember has 50% of wild type and 50% of random amino acid residues, thispercentage or ratio (1:1) can be varied in such a manner that only oneamino acid or up to 90% of a wild type amino acid sequence can bereplaced by random amino acid residues.

The complexities of VELs can range from a VEL composed of 20 epitopevariants where only one wild-type amino acid residue is replaced in theepitope by a random amino acid (e.g., 20 total peptides in the VEL), andup to about 208 epitope variants, where several amino acid residues aremutated. In some embodiments, the complexities of VELs can range fromabout 20 different amino acids to about 2010 different amino acids,depending on the number of variable amino acids, as one of skill in theart would recognize and understand based on the present disclosure andcommon knowledge. Further, the appearance of any amino acid other thanwild type amino acid within the epitopes derived from geneticallyvariable pathogens or disease-related antigens can include a pathogen,for example, including, but not limited to, Human Immunodeficiency Virus(HIV), Simian Immunodeficiency Virus (SIV), Hepatitis A, Hepatitis B,Hepatitis C, rhinovirus, influenza virus, Plasmodium falciparum,tuberculosis, cancer (e.g., breast cancer), and infections salmon anemiavirus (ISAV), or some tumor antigens, where this event can be a frequentphenomenon. A VEL-based immunogen construction can represent antigenicdiversity observed during the infection with the above mentionedpathogens and/or in diseases. Use of VEL immunogens as disclosed hereinpermits the generation of novel prophylactic and therapeutic vaccinescapable of inducing a broad range of protective immune responses beforethe appearance of mutated epitopes (before infection) or when theamounts of mutated epitopes are low (early stages of infection and/ordisease progression). VELs and VEL compositions can be usedprophylactically and/or therapeutically to treat, prevent, and/or reducethe risk of developing various diseases from various pathogens, such asa cancerous tumor.

VELs can be generated based on defined pathogen or disease-relatedantigen-derived cytotoxic T lymphocyte (CTL), helper T lymphocyte (Th)or B lymphocyte epitopes and additionally, on epitopes derived fromantigenically variable or relatively conserved regions of protein.Alternatively, VELs can be generated based on up to 50 amino acid longpeptide regions of antigens containing clusters of epitopes. Anindividual VEL can contain: [1] variants of one CTL, Th or B cellepitope; [2] variants of several different CTL, Th or B cell epitopes;[3] any combination of these mutated CTL, Th and B cell epitopesexpressed in a single up to 120 amino acid long artificial recombinantpolypeptide; [4] up to 50 amino acid long mutated wild type-relatedpeptide carrying several CTL, Th and/or B cell epitopes. Additionally,the VELs can be generated based on 7-50 amino acid peptides selectedfrom antigenically variable or relatively conserved regions of pathogen-or disease-related proteins without a prior knowledge of the existenceof epitopes in these peptide regions. Candidate epitopes can be selectedfrom scientific literature or from public databases. It may be useful toinclude CTL epitopes in VELs, since the escape from protective CTLresponses is an important mechanism for immune evasion by manypathogens, for example HIV and SIV.

VELs can take the form of DNA constructs, recombinant polypeptides orsynthetic peptides and can be generated using standard molecular biologyor peptide synthesis techniques, as discussed below. For example togenerate a DNA fragment encoding particular epitope variants bearingpeptides, a synthetic 40-70 nucleotide (nt) long oligonucleotide (oligo)carrying one or more random amino acid-coding degenerate nucleotidetriplet(s) may be designed and produced. The epitope-coding region ofthis oligo (oligol) may contain non-randomized 9-15 nt segments at 5′and 3′ flanking regions that may or may not encode naturalepitope-flanking 3-5 amino acid residues. Then, 2 oligos that overlap at5′ and 3′ flanking regions of oligol and carry nt sequences recognizedby hypothetical restriction enzymes A and B, respectively, may besynthesized and after annealing reaction with oligol used in a PCR. ThisPCR amplification will result in mutated epitope library-encoding DNAfragments that after digestion with A and B restriction enzymes may becombined in a ligation reaction with corresponding bacterial, viral oreukaryotic cloning/expression vector DNA digested with the same enzymes.Ligation mixtures can be used to transform bacterial cells to generatethe VEL and then expressed as a plasmid DNA construct, in a mammalianvirus or as a recombinant polypeptide. This DNA can also be cloned inbacteriophage, bacterial or yeast display vectors, allowing thegeneration of recombinant microorganisms.

In a similar manner, DNA fragments bearing 20-200 individual nucleotidescan encode various combinations of different mutated epitope variants.These nucleic acid molecules can be created using sets of longoverlapping oligos and a pair of oligos carrying restriction enzymerecognition sites and overlapping with adjacent epitope-coding oligos at5′ and 3′flanking regions. These oligos can be combined, annealed andused in a PCR assembly and amplification reactions. The resulting DNAsmay be similarly cloned in the above mentioned vectors.

In another embodiment, nucleic acid sequence molecules encoding mutatedepitope clusters may also be obtained using pairs of wild typesequence-specific oligos carrying DNA restriction sites and pathogen- orantigen-derived genomic or cDNA as template in a PCR with an error-proneDNA polymerase. These DNAs also may be cloned in corresponding vectors.The VELs may be expressed in mammalian virus vectors, such as modifiedVaccinia ankara, an adenoviral, a canary pox vectors, produced asrecombinant polypeptides or as recombinant microorganisms and usedindividually as immunogens or may be combined and used as a mixture ofVELs.

In one example, synthetic peptide VELs varying in length from 7 to 50amino acid residues may be generated by solid phase Fmoc peptidesynthesis technique where in a coupling step equimolar mixtures of allproteogeneic amino acid residues may be used to obtain randomized aminoacid positions. This technique permits the introduction of one or morerandomized sequence positions in selected epitope sequences and thegeneration of VELs with complexities of up to 10⁹.

Immunogens based on VELs can be useful for inducing protective immuneresponses against pathogens and tumors with antigenic variability, suchas cancer, as well as may be effective in modulating allergy,inflammatory and autoimmune diseases. In one embodiment, vaccinecompositions containing one or more VELs may be formulated with apharmaceutically acceptable carrier, excipient and/or adjuvant, andadministered to a subject, such as a non-human animal or a humanpatient. Compositions containing VELs comprising peptides can beadministered to a subject, such as a human, therapeutically orprophylactically at dosages ranging from about 100 m to about 1 mg ofisolated peptides. Compositions containing VELs including nucleic acidsequences can be administered to a subject, such as a human,therapeutically or prophylactically at dosages ranging from about 1×10¹⁰to about 5×10¹⁵ CFU of bacteriophage particles. In some embodiments,VELs administered to a human subject can reduce onset of a disease suchas a cancer (e.g., a malignant cancer such as a malignant tumorinvolving survivin) and/or VELs administered to a human subject cantreat a disease already existing in the human subject (e.g., a cancerousmalignancy involving survivin). Other approaches for the construction ofVELs, expression and/or display vectors, optimum vaccine composition,routes for vaccine delivery and dosing regimens capable of inducingprophylactic and therapeutic benefits may be determined by one skilledin the art based on the present disclosure. For example, compositionscontaining VELs can be administered to a subject as a single doseapplication, as well as a multiple dose (e.g., booster) application.Multiple dose applications can include, for example, administering fromabout 1 to about 25 total dose applications, with each dose applicationadministered at one or more dosing intervals that can range from about 7days to about 14 days (e.g., weekly). In some embodiments, dosingintervals can be administered daily, two times daily, twice weekly,weekly, monthly, bi-monthly, annually, or bi-annually, depending on theparticular needs of the subject and the characteristics of the conditionbeing treated or prevented (or reducing the risk of getting thecondition), as would be appreciated by one of skill in the art based onthe present disclosure.

The skilled artisan will realize that in alternative embodiments, lessthan the 20 naturally occurring amino acids may be used in arandomization process. For example, certain residues that are known tobe disruptive to protein or peptide secondary structure, such as prolineresidues, may be less preferred for the randomization process. VELs maybe generated with the 20 naturally occurring amino acid residues or withsome subset or derivatives of the 20 naturally occurring amino acidresidues. In various embodiments, in addition to or in place of the 20naturally occurring amino acid residues, the VELs may contain at leastone modified amino acid, including but not limited to, those presentedon Table 1 below.

TABLE 1 Modified amino acid residues Abbr. Amino Acid Aad 2-Aminoadipicacid Baad 3-Aminoadipic acid Bala β-alanine, β-Amino-propionic acid Abu2-Aminobutyric acid 4Abu 4-Aminobutyric acid, piperidinic acid Acp6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib 2-Aminoisobutyric acidBaib 3-Aminoisobutyric acid Apm 2-Aminopimelic acid Dbu2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelic acid Dpr2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsn N-EthylasparagineHyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp 3-Hydroxyproline 4Hyp4-Hydroxyproline Ide Isodesmosine AIle allo-Isoleucine MeGlyN-Methylglycine, sarcosine MeIle N-Methylisoleucine MeLys6-N-Methyllysine MeVal N-Methylvaline Nva Norvaline Nle Norleucine OrnOrnithine

VELs may be made by any technique known to those of skill in the art,including the expression of polypeptides or peptides through standardmolecular biological techniques or the chemical synthesis of peptides.The nucleotide and polypeptide and peptide sequences corresponding tovarious pathogen- or disease-related antigens are known in the art andmay be found at computerized databases known to those of ordinary skillin the art. One such database is the National Center for BiotechnologyInformation's Genbank and GenPept databases. Any such known antigenicsequence may be used in the practice of the claimed methods andcompositions.

Combinatorial Libraries

Combinatorial libraries of such compounds or of such targets can becategorized into three main categories. The first category relates tothe matrix or platform on which the library is displayed and/orconstructed. For example, combinatorial libraries can be provided (i) ona surface of a chemical solid support, such as microparticles, beads ora flat platform; (ii) displayed by a biological source (e.g., bacteriaor phage); and (iii) contained within a solution. In addition, threedimensional structures of various computer generated combinatorialmolecules can be screened via computational methods.

Combinatorial libraries can be further categorized according to the typeof molecules represented in the library, which can include, (i) smallchemical molecules; (ii) nucleic acids (DNA, RNA, etc.); (iii) peptidesor proteins; and (iv) carbohydrates.

The third category of combinatorial libraries relates to the method bywhich the compounds or targets are synthesized, such synthesis istypically effected by: (i) in situ chemical synthesis; (ii) in vivosynthesis via molecular cloning; (iii) in vitro biosynthesis by purifiedenzymes or extracts from microorganisms; and (iv) in silico by dedicatedcomputer algorithms.

Combinatorial libraries indicated by any of the above synthesis methodscan be further characterized by: (i) split or parallel modes ofsynthesis; (ii) molecules size and complexity; (iii) technology ofscreening; and (iv) rank of automation in preparation/screening.

The complexity of molecules in a combinatorial library depends upon thediversity of the primary building blocks and possible combinationsthereof. Furthermore, several additional parameters can also determinethe complexity of a combinatorial library. These parameters include (i)the molecular size of the final synthesis product (e.g., oligomer orsmall chemical molecule); (ii) the number of bonds that are created ineach synthesis step (e.g., one bond vs. several specific bonds at atime); (iii) the number of distinct synthesis steps employed; and (iv)the structural complexity of the final product (e.g., linear vs.branched molecules).

Combinatorial libraries can be synthesized of several types of primarymolecules, including, but not limited to, nucleic and amino acids andcarbohydrates. Due to their inherent single bond type complexity,synthesizing nucleic and amino acid combinatorial libraries typicallynecessitates only one type of synthesis reaction. On the other hand, dueto their inherent bond type complexity, synthesizing complexcarbohydrate combinatorial libraries necessitates a plurality ofdistinct synthesis reactions.

Expression of Proteins or Peptides

In certain embodiments, it may be preferred to make and use anexpression vector that encodes and expresses a particular VEL. Genesequences encoding various polypeptides or peptides may be obtained fromGenBank and other standard sources, as disclosed above. Expressionvectors containing genes encoding a variety of known proteins may beobtained from standard sources, such as the American Type CultureCollection (Manassas, Va.). For relatively short VELs, it is within theskill in the art to design synthetic DNA sequences encoding a specifiedamino acid sequence, using a standard codon table, as discussed above.Genes may be optimized for expression in a particular species of hostcell by utilizing well-known codon frequency tables for the desiredspecies. Genes may represent genomic DNA sequences, containing bothintrons and exons, or more preferably comprise cDNA sequences, withoutintrons.

Regardless of the source, a coding DNA sequence of interest can beinserted into an appropriate expression system. The DNA can be expressedin any number of different recombinant DNA expression systems togenerate large amounts of the polypeptide product, which can then bepurified and used in various embodiments of the present disclosure.

Examples of expression systems known to the skilled practitioner in theart include bacteria such as E. coli, yeast such as Pichia pastoris,baculovirus, and mammalian expression systems such as in Cos or CHOcells. Expression is not limited to single cells, but may also includeprotein production in genetically engineered transgenic animals, such asrats, cows or goats. A complete gene can be expressed or, alternatively,fragments of the gene encoding portions of polypeptide can be produced.

In certain broad applications of the disclosure, the sequence encodingthe polypeptide may be analyzed to detect putative transmembranesequences. Such sequences are typically very hydrophobic and are readilydetected by the use of standard sequence analysis software, such asMacVector (IBI, New Haven, Conn.). The presence of transmembranesequences may be deleterious when a recombinant protein is synthesizedin many expression systems, especially E. coli, as it leads to theproduction of insoluble aggregates which are difficult to renature intothe native conformation of the protein. Deletion of transmembranesequences typically does not significantly alter the conformation of theremaining protein structure. Deletion of transmembrane-encodingsequences from the genes used for expression can be achieved by standardtechniques. For example, fortuitously-placed restriction enzyme sitescan be used to excise the desired gene fragment, or PCR-typeamplification can be used to amplify only the desired part of the gene.

The gene or gene fragment encoding a polypeptide may be inserted into anexpression vector by standard subcloning techniques. An E. coliexpression vector may be used which produces the recombinant polypeptideas a fusion protein, allowing rapid affinity purification of theprotein. Examples of such fusion protein expression systems are theglutathione S-transferase system (Pharmacia, Piscataway, N.J.), themaltose binding protein system (NEB, Beverley, Mass.), the FLAG system(IBI, New Haven, Conn.), and the 6× His system (Qiagen, Chatsworth,Calif.).

Some of these systems produce recombinant polypeptides bearing only asmall number of additional amino acids, which are unlikely to affect theactivity or binding properties of the recombinant polypeptide. Forexample, both the FLAG system and the 6× His system add only shortsequences, both of which have no adverse effect on folding of thepolypeptide to its native conformation. Other fusion systems aredesigned to produce fusions wherein the fusion partner is easily excisedfrom the desired polypeptide. In one embodiment, the fusion partner islinked to the recombinant polypeptide by a peptide sequence containing aspecific recognition sequence for a protease. Examples of suitablesequences are those recognized by the Tobacco Etch Virus protease (LifeTechnologies, Gaithersburg, Md.) or Factor Xa (New England Biolabs,Beverley, Mass.).

The expression system used may also be one driven by the baculoviruspolyhedron promoter. The gene encoding the polypeptide may bemanipulated by standard techniques in order to facilitate cloning intothe baculovirus vector. One baculovirus vector is the pBlueBac vector(Invitrogen, Sorrento, Calif.). The vector carrying the gene for thepolypeptide is transfected into Spodoptera frugiperda (Sf9) cells bystandard protocols, and the cells are cultured and processed to producethe recombinant protein.

To express a recombinant encoded protein or peptide, whether mutant orwild-type, one would prepare an expression vector that comprises one ofthe isolated nucleic acids under the control of, or operatively linkedto, one or more promoters. To bring a coding sequence “under the controlof” a promoter, one positions the 5′ end of the transcription initiationsite of the transcriptional reading frame generally from about 1 toabout 50 nucleotides “downstream” (3′) of the chosen promoter. The“upstream” promoter stimulates transcription of the DNA and promotesexpression of the encoded recombinant protein.

Many standard techniques are available to construct expression vectorscontaining the appropriate nucleic acids andtranscriptional/translational control sequences in order to achieveprotein or peptide expression in a variety of host-expression systems.Cell types available for expression include, but are not limited to,bacteria, such as E. coli and B. subtilis transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectors.

Certain examples of prokaryotic hosts are E. coli strain RR1, E. coliLE392, E. coli B, E. coli X 1776 (ATCC No. 31537) as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325); bacilli such asBacillus subtilis; and other enterobacteriaceae such as Salmonellatyphimurium, Serratia marcescens, and various Pseudomonas species.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli is oftentransformed using pBR322, a plasmid derived from an E. coli species.pBR322 contains genes for ampicillin and tetracycline resistance andthus provides easy means for identifying transformed cells. The pBRplasmid, or other microbial plasmid or phage must also contain, or bemodified to contain, promoters which may be used by the microbialorganism for expression of its own proteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism may be used astransforming vectors in connection with these hosts. For example, thephage lambda GEMTM-11 may be utilized in making a recombinant phagevector which may be used to transform host cells, such as E. coli LE392.

Further useful vectors include pIN vectors and pGEX vectors, for use ingenerating glutathione S transferase (GST) soluble fusion proteins forlater purification and separation or cleavage. Other suitable fusionproteins are those with B galactosidase, ubiquitin, or the like.

Promoters that are most commonly used in recombinant DNA constructioninclude the β-lactamase (penicillinase), lactose and tryptophan (trp)promoter systems. While these are the most commonly used, othermicrobial promoters have been discovered and utilized, and detailsconcerning their nucleotide sequences have been published, enablingthose of skill in the art to ligate them functionally with plasmidvectors.

For expression in Saccharomyces, the plasmid YRp7, for example, iscommonly used. This plasmid already contains the trpl gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1. Thepresence of the trpl lesion as a characteristic of the yeast host cellgenome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other suitable promoters, which have the additional advantage oftranscription controlled by growth conditions, include the promoterregion for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization.

In addition to micro-organisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. In addition to mammalian cells, these include insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus); and plant cell systems infected with recombinant virusexpression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaicvirus, TMV) or transformed with recombinant plasmid expression vectors(e.g., Ti plasmid) containing one or more coding sequences.

In a useful insect system, Autographa californica nuclear polyhedrosisvirus (AcNPV) is used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The isolated nucleic acid codingsequences are cloned into non-essential regions (e.g., polyhedrin gene)of the virus and placed under control of an AcNPV promoter (e.g.,polyhedrin promoter). Successful insertion of the coding sequencesresults in the inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (e.g., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed.

Examples of useful mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cell lines, W138, BHK, COS-7, 293, HepG2,3T3, RIN and MDCK cell lines. In addition, a host cell strain may bechosen that modulates the expression of the inserted sequences, ormodifies and processes the gene product in the specific fashion desired.Such modifications (e.g., glycosylation) and processing (e.g., cleavage)of protein products may be important for the function of the encodedprotein.

Different host cells have characteristic and specific mechanisms for thepost-translational processing and modification of proteins. Appropriatecells lines or host systems may be chosen to ensure the correctmodification and processing of the foreign protein expressed. Expressionvectors for use in mammalian cells ordinarily include an origin ofreplication (as necessary), a promoter located in front of the gene tobe expressed, along with any necessary ribosome binding sites, RNAsplice sites, polyadenylation site, and transcriptional terminatorsequences. The origin of replication may be provided either byconstruction of the vector to include an exogenous origin, such as maybe derived from SV40 or other viral (e.g., Polyoma, Adeno, VSV, BPV)source, or may be provided by the host cell chromosomal replicationmechanism. If the vector is integrated into the host cell chromosome,the latter is often sufficient.

The promoters may be derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter) as known inthe art.

A number of viral based expression systems may be utilized, for example,commonly used promoters are derived from polyoma, Adenovirus 2, and mostfrequently Simian Virus 40 (SV40). The early and late promoters of SV40virus are useful because both are obtained easily from the virus as afragment which also contains the SV40 viral origin of replication.Smaller or larger SV40 fragments may also be used, provided there isincluded the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the viral origin of replication.

In one example where an adenovirus is used as an expression vector, thecoding sequences may be ligated to an adenovirustranscription/translation control complex (e.g., the late promoter andtripartite leader sequence). This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingproteins in infected hosts.

Specific initiation signals known in the art may also be required forefficient translation of the claimed isolated nucleic acid codingsequences. One of ordinary skill in the art would readily be capable ofdetermining this and providing the necessary signals.

In eukaryotic expression, one will also typically desire to incorporateinto the transcriptional unit an appropriate polyadenylation site if onewas not contained within the original cloned segment. Typically, thepoly A addition site is placed about 30 to 2000 nucleotides “downstream”of the termination site of the protein at a position prior totranscription termination. For long-term, high-yield production ofrecombinant proteins by stable expression known in the art may berequired.

A number of selection systems may be used, including but not limited to,the herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase and adenine phosphoribosyltransferase genes,in tk-, hgprt- or aprt-cells, respectively. Also, antimetaboliteresistance may be used as the basis of selection for dhfr, that confersresistance to methotrexate; gpt, that confers resistance to mycophenolicacid; neo, that confers resistance to the aminoglycoside G-418; andhygro, that confers resistance to hygromycin. These and other selectiongenes may be obtained in vectors from, for example, ATCC or may bepurchased from a number of commercial sources known in the art (e.g.,Stratagene, La Jolla, Calif.; Promega, Madison, Wis.).

Where substitutions into naturally occurring pathogen- ordisease-related polypeptide sequences are desired, the nucleic acidsequences encoding the native polypeptide sequence may be manipulated bywell-known techniques, such as site-directed mutagenesis or by chemicalsynthesis of short oligonucleotides followed by restriction endonucleasedigestion and insertion into a vector, by PCR based incorporationmethods, or any similar method known in the art.

Protein Purification

In certain embodiments a polypeptide or peptide may be isolated orpurified. Protein purification techniques are well known to those ofskill in the art. These techniques involve, at one level, thehomogenization and crude fractionation of the cells, tissue or organ topolypeptide and non-polypeptide fractions. The peptide or polypeptide ofinterest may be further purified using chromatographic andelectrophoretic techniques to achieve partial or complete purification(or purification to homogeneity). Analytical methods well suited to thepreparation of a pure peptide are ion-exchange chromatography, gelexclusion chromatography, polyacrylamide gel electrophoresis, affinitychromatography, immunoaffinity chromatography and isoelectric focusing.An efficient method of purifying peptides is fast performance liquidchromatography (FPLC) or even HPLC.

A purified polypeptide or peptide is intended to refer to a composition,isolatable from other components, wherein the polypeptide or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified polypeptide or peptide, therefore, also refers to apolypeptide or peptide free from the environment in which it maynaturally occur. Generally, “purified” will refer to a polypeptide orpeptide composition that has been subjected to fractionation to removevarious other components. Where the term “substantially purified” isused, this designation will refer to a composition in which thepolypeptide or peptide forms the major component of the composition,such as constituting about 50%, about 60%, about 70%, about 80%, about90%, about 95%, or more of the polypeptides in the composition. Variousmethods for quantifying the degree of purification of the polypeptide orpeptide are known to those of skill in the art in light of the presentdisclosure. These include, for example, assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis.

Various techniques suitable for use in protein purification arecontemplated herein and are well known. There is no general requirementthat the polypeptide or peptide always be provided in their mostpurified state. Indeed, it is contemplated that less substantiallypurified products will have utility in certain embodiments. In anotherembodiment, affinity chromatography may be required and any means knownin the art is contemplated herein.

Formulations and Routes for Administration to Subjects

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions (e.g., VEL vaccine compositions) ina form appropriate for the intended application. Generally, this willentail preparing compositions that are essentially free of impuritiesthat could be harmful to human or animal subjects.

One generally will desire to employ appropriate salts and buffers torender polypeptides stable and allow for uptake by target cells. Aqueouscompositions may comprise an effective amount of polypeptide dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.Such compositions also are referred to as innocula. The phrase“pharmaceutically or pharmacologically acceptable” refers to molecularentities and compositions that do not produce adverse, allergic, orother untoward reactions when administered to an animal or a human. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the polypeptides of the present disclosure, its use intherapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure will be via any common route so longas the target tissue is available via that route. This includes oral,nasal, buccal, rectal, vaginal or topical. Alternatively, administrationmay be by orthotropic, intradermal, subcutaneous, intramuscular,intraperitoneal, intraarterial or intravenous injection. Suchcompositions normally would be administered as pharmaceuticallyacceptable compositions, as described above.

The active compounds also may be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersionsalso can be prepared in glycerol, liquid polyethylene glycols, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In certain examples, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. Regarding sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

The compositions of the present disclosure may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion. Some variation in dosage will necessarilyoccur depending on the condition of the subject being treated. Theperson responsible for administration will, in any event, determine theappropriate dose for the individual subject. Moreover, for humanadministration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

The VELs and VEL compositions of the present disclosure may also be usedin conjunction with targeted therapies, including but not limited to,therapies designed to target tumors and the cells underlying the tumor.Many different targeted therapies have been approved for use in cancertreatment. For example, these therapies can include hormone therapies,signal transduction inhibitors, gene expression modulator, apoptosisinducer, angiogenesis inhibitor, immunotherapies, and toxin deliverymolecules. Additionally, cancer vaccines and gene therapy can beconsidered targeted therapies because they interfere with the growth ofspecific cancer cells (e.g., breast cancer cells).

EXAMPLES

Embodiments of the present disclosure are included to demonstratecertain embodiments presented herein. It should be appreciated by thoseof skill in the art that the techniques disclosed in the examples thatfollow represent techniques discovered to function well in the practicesdisclosed herein. However, those of skill in the art should, in light ofthe present disclosure, appreciate that many changes can be made in thecertain embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope herein.

Example 1 Construction of Variable Epitope Libraries (VELs)

In order to avoid tumor escape, it is desirable to target a tumorantigen that is essential for tumor survival and expressed by tumors athigh levels. One of these antigens is survivin, an oncogenicinhibitor-of-apoptosis protein, which is expressed at high levels invirtually all malignancies and is commonly referred to as a universaltumor antigen. Additionally, survivin-specific T-cell reactivitystrongly correlates with tumor response and subject survival. In oneembodiment of the present disclosure, phage display VELs and syntheticpeptide VELs were generated based on the survivin-derived CTL epitopepresented below:

-   GWXPXDXPI (SEQ ID NO: 1), where X is any of the 20 naturally    occurring amino acids or derivatives thereof.

VELs were generated using the recombinant M13 phage display system basedon the survivin-derived H-2Dd-restricted wild-type CTL epitope,GWEPDDNPI (SEQ ID NO: 2), referred to as SWT. The recombinant phagedisplay library comprising the wild-type survivin epitope is referred toas FSWT, and the recombinant phage display VEL comprising the variableepitopes of wild-type survivin is referred to as FSVL (FIG. 1A).Additionally, the synthetic peptide library comprising the wild-typesurvivin epitope is referred to as PSWT, and the synthetic peptide VELcomprising the variable epitopes of wild-type survivin is referred to asPSVL (FIG. 1A). The epitope variants comprising the combinatorial VELs,were generated using degenerate oligonucleotides encoding a library ofepitope variants with structural composition GWXPXDXPI, (SEQ ID NO: 1),where X is any of 20 natural amino acids (SEQ ID NO: 1). The DNAfragments corresponding to the SWT and SVL, respectively, were amplifiedby PCR and cloned into pG8SAET phagemid vector that allows theexpression of epitopes at high copy numbers as peptides fused to phagecpVIII. The amino acids at the MHC-binding anchor positions weremaintained within the epitope, while mutations were introduced atpositions responsible for interaction with TCR. Thus, each variantepitope has random amino acid substitutions (mutations) at 3 definedpositions within the SWT and, the theoretical complexity of such libraryis 8×103 individual members. The FSVL phage library has a complexity of10,500 original clones.

As described above, VELs can also be generated based on other epitopesfound within the full length survivin peptide. For example, VELs can begenerated based on the epitope presented below:

(SEQ ID NO: 3) AGFIHCPTENEPDLAQXFFXFKELEGWXPXDXPIEEHXKHSXGCAFLX

where X is any one of the 20 naturally occurring amino acids orderivatives thereof.

The various sequences for the libraries described above were verifiedand a subset was generated to T-cell assays and a panel of epitopes forin vivo studies. Twenty-six phage clones were randomly isolated from theFSVL, and the amino acid sequences of the correspondingpeptides/epitopes were determined after DNA sequencing. As presented inTable 2, 12-16 different amino acids were detected at each respectivevariable amino acid position in the 26 epitope variants, indicating anacceptable level of epitope diversity. In order to generate a panel ofvariant epitopes for in vivo studies, 87 phage clones were randomlyisolated from the FSVL epitope library, and used in T-cell assays. Also,a non-related phage clone FB22 was used as the control in immunizationexperiments.

Example 2 Anti-Tumor Effects in Mice Vaccinated with VELs

An analysis of the immunogenic properties of VEL-based vaccinecompositions before (e.g., prophylactic treatment) and after (e.g.,therapeutic treatment) tumor challenge was performed. BALB/c mice werechallenged by exposure to syngeneic 4T1 tumor cell. Groups of mice wereimmunized with FSVL, FSWT or FB22 control phages expressing VEL-basedepitope variants, wild-type SWT epitopes, or the control non-related B22epitopes, respectively, by intravenous (iv) injection as illustratedschematically in FIG. 1 . As a control, a group of non-immunized micewas included. Forty days after vaccination, all mice were inoculatedwith 104 4T1 tumor cells and monitored prospectively for development oftumors (FIG. 1B). As demonstrated in FIG. 2A, a statisticallysignificant (P<0.05) tumor growth inhibition in a prophylactic settingwas observed in FSVL-vaccinated mice compared with FSWT-vaccinated mice,and controls (e.g., FB22-vaccinated mice and control non-immunized micereceiving only a 4T1 cell transplant).

To test the anti-tumor effects of VELs on already established tumors, amouse model was used. Mice were transplanted with 4T1 cells andvaccinated with FSVL, FSWT and FB22 phages, or PSWT (synthetic peptidecorresponding to SWT epitope GWEPDDNPI) (SEQ ID NO: 2), PSVL (syntheticpeptides corresponding to the SVL (GWXPXDXPI) (SEQ ID NO: 1),peptide/epitope library) and PG5D (a control peptide/epitope library(A[G/F]PXXXXX[L/M]), by single i.v. and s.c. injection (FIG. 2B and FIG.2D, respectively), on day 5 after tumor challenge, as illustratedschematically in FIG. 1C. Inhibition of tumor growth was demonstrated inFSVL-vaccinated mice (FIG. 2B, single injection) and in PSVL-vaccinatedmice (FIG. 2D, single injection), as compared to mice immunized withFSWT, PSWT, or non-immunized tumor-bearing mice.

Single vaccinations of mice with phage and synthetic peptide VELs ondays 14 and 21 after tumor inoculation was not inhibitory for tumorgrowth (FIG. 1C). However, multiple immunizations (e.g., boosters)enhanced anti-tumor effects. As illustrated in FIG. 2C, three boosterimmunizations with FSVL improved the vaccine potency as compared with asingle priming dose of FSVL (FIG. 2B), resulting in statisticallysignificant tumor growth inhibition. However, three boosterimmunizations of mice according to the same schedule with syntheticpeptide VELs did not lead to as significant of an increase in vaccineefficacy (FIG. 2E) as compared to a single priming dose (FIG. 2D). Nosignificant anti-tumor effect was observed in mice bearing established(>10 mm2, day 14) or large (>40 mm2, day 21) tumors that were primed ondays 14 or 21, and then received one or two booster immunizations ondays 17 and 23 or on day 26, respectively, by phage or syntheticpeptides (FIG. 1D). The immunized mice undergoing necropsy were alsoroutinely assessed for evidence of autoimmunity; no abnormal lymphocyticinfiltrates into organs were observed.

These data indicate that vaccination with VELs in the form ofrecombinant M13 phage particles and synthetic peptides has statisticallysignificant anti-tumor effects when applied prophylactically (FIG. 2A)and therapeutically (FIGS. 2B-2E) in a mouse 4T1 mammary carcinomamodel.

Example 3 Cellular Immune Responses Induced by VELs

Immunization with FSVL phage-displayed VELs were able to elicit immuneresponses to CTL epitope variants as indicated with T-cell proliferationassays. Mice were immunized by a single injection with FSVL or FSWT, anda subset of these mice were challenged with 4T1 cells 40 days later,while the rest of the mice were not challenged with tumor cells. Fifteendays post-challenge, splenocytes were prepared from both groups,stimulated in vitro using the panel of 87 phage clones, and the breadth(e.g., number of responding epitope variants) and the magnitude of totalT-cell proliferative responses were measured by flow cytometry. To moreclearly visualize differential recognition of epitope variants by thespleen cells of mice immunized with FSVL or FSWT, differences observedbetween experimental groups were calculated, and illustrated in FIGS.3A-3D.

Immunization with the FSVL library generated a pool of splenocytes withhighly variable capacities to recognize individual epitope variants(see, e.g., FIG. 3A). The levels of proliferation of the splenocytesfrom mice immunized with FSVL and FSWT were from about 8.50-43.60% toabout 9.61-28.41%, respectively (p<0.006) (FIG. 3A). Additionally, 29out of 87 epitope variants (or 33%) showed higher stimulatory capacityagainst cells from FSVL-immunized mice compared with FSWT-immunized mice(3-20% increase in percentages of cell proliferation). Only 14 epitopevariants showed about a 3-11% increase in stimulatory capacity againstcells from FSWT-immunized mice (FIG. 3A; 29/87 as compared to 14/87;p<0.009). These data indicate the superior immunogenicity of FSVL overFSWT and the induction of long-lasting immune memory. Additionally, 18epitope variants were more immunogentic than the wild-typeepitope-expressing FSWT phage. The FSWT phage elicited a more thantwo-fold stronger immunogenic response against splenocytes from both theFSVL- and FSWT-immunized mice as compared to the FB22 control phage. TheFB22 control phage produced only background levels of cell proliferation(data not shown).

After tumor challenge (e.g., therapeutic treatment), however, a stronginhibition of these immune responses was observed (FIG. 3B). Epitopevariants were able to stimulate cells from mice immunized with FSWT moreefficiently than cells from FSVL-immunized mice (59/87 as compared to3/87 p<0.0001). After tumor challenge, both FSWT and FB22 controlsinduced similar background level of cell proliferation against spleencells from either FSVL- or FSWT-immunized mice (data not shown). Theobserved pattern of epitope recognition was changed after tumorchallenge leading to general inhibition of cell proliferation and topreferential recognition by variant epitopes of spleen cells fromFSWT-immunized mice compared with FSVL-immunized animals (FIG. 3B).

The ability of the FSVL phage-display VEL to elicit immune responses toCTL epitope variants in therapeutic setting was also tested. Groups ofmice were immunized by a single injection with FSVL, FSWT or the controlFB22 phage on day 5 after tumor challenge, and a subset of mice wereleft naïve to any treatment (FIG. 3C). Fifteen days after the tumorchallenge, splenocytes were prepared, stimulated in vitro with the panelof 87 phage clones, and analyzed using the T-cell proliferation assay.As illustrated in FIG. 3C, the general levels of proliferation of cellsfrom FSVL- and FSWT-immunized mice was lower (with absolute values offrom about 6.86-28.46% to about 12.14-27.64%, respectively; p<0.0001)than those obtained in prophylactic study (FIG. 3B). Cells fromFSVL-immunized mice exhibited proliferative responses to 53 epitopevariants (about 61% of variants) with values from about 3-10% abovethose obtained with cells from FSWT-immunized mice (53/87 as compared to2/87; p<0.0001). The spleen cells from tumor-bearing mice immunized withthe control FB22 phage demonstrated only background levels of cellproliferation against almost all variant epitopes (data not shown)indicating the induction of epitope variant- and epitope-specific immuneresponses by the VEL-bearing FSVL. In the presence of an establishedtumor in an acceptable mouse model, vaccination with FSVL producedsuperior immunogenicity compared to vaccination with FSWT. The responsesagainst individual epitope variants were highly reproducible and fromabout 11 to about 18 epitope variants resulted in a stronger immuneresponse than the FSWT wild-type epitope against cells from FSVL- andFSWT-immunized mice, respectively (data not shown). These data indicatethat in both prophylactic and therapeutic settings, several epitopevariants are more potent immunogens than the wild-type CTL epitope.

The antigenic properties of the generated panel of epitope variantsagainst spleen cells obtained from non-immunized control mice was alsotested in order to measure a background level of immune recognition andto determine the changes induced in epitope reactivity profile aftertumor challenge. While the general level of proliferation of splenocytesfrom both groups of mice were similar (from about 10.49-37.79% to about11.73-37.73%, respectively; p<0.309), the spontaneous cell-stimulatingcapacities of 27 epitope variants (or 31%) were diminished after tumorchallenge, as indicated by decreased percentages of cell proliferation(differences between groups were about 3-18%), as illustrated in FIG.3D. Additionally, 17 epitope variants (or 19%) were preferentiallyactivating splenocytes from tumor-bearing mice as compared with controlmice (about 3-21% of differences; 27/87 as compared to 17/87; p<0.083).Again, as in the example of tumor challenge of immunized mice describedabove, neither FSWT nor the FB22 controls stimulated splenocyteproliferation from naïve or tumor-inoculated mice.

Together, these data demonstrate that vaccination with VELs based on asurvivin-derived CTL epitope induces specific anti-tumor cellular immuneresponses and suggest the superiority of VEL-based immunogens overimmunogens carrying defined wild-type epitope in their capacity toinduce broad and potent immune responses without inducing harmfulautoimmune reactions.

Example 4 Phenotypic Analysis of Activated Lymphocytes

Flow cytometry and intracellular cytokine staining (ICS) assays wereperformed to determine which subpopulations of T-cells wereproliferating in the T-cell proliferation assays, and to test whetherimmunization with VELs induces epitope-specific activation of bothCD4+IFN-γ+ and CD8+IFN-γ+ cells (FIGS. 4A-4D). Pooled splenocytes fromeach group of mice used in prophylactic and therapeutic vaccineexperiments of FIGS. 3A-3D were analyzed by FACS either after 6 hours ofstimulation (ex vivo cells) or upon 3 days of incubation with 9phage-displayed variant epitopes selected at random among 29 clones thatshowed superior antigenic properties in the T-cell proliferation assaysdescribed above, as well as with the FSWT wild-type epitope and the FB22control epitope.

In the ICS assays with ex vivo cells in a therapeutic vaccine setting,while the spleen cells collected from mice immunized with FSVL containedonly small proportion of IFN-γ-producing CD8+ T cells (about0.05-0.25%), a several-fold increase in the percentages of epitopevariant-specific CD8+IFN-γ+ cells was observed upon stimulation withseveral clones during a 72 hr. period (48-fold increase for clone 27;14-fold increase for clone 45; 7-fold increase for clone 58; 6.7-foldincrease for clone 73; and a 21-fold increase for clones 82) (FIG. 4A).The incubation of spleen cells with the rest of the epitope variants aswell as with the FB22 control epitope resulted in less than about 1% ofIFN-γ-producing cells (FIG. 4B). In the example using CD4+IFN-γ+ cells,only clones 27 and 45 were stimulatory against cells from FSVL-immunizedmice, exhibiting a 2-3 fold increase in cell percentages.

In a prophylactic setting, the presence of epitope variant-specificCD8+IFN-γ+ ex vivo cells in FSVL-immunized mice was detected in clones45, 53, 58 and 80, all of which showed cell percentages above thoseobtained with the cells from mice immunized with FSWT, FB22 controls,and untreated mice (FIG. 4C). Incubation for 72 hrs. led only to slightincreases in cell proliferation, and cell numbers decreased with severalepitopes (clones 53, 58, 73 and 80) (FIG. 4D).

Together, these results demonstrate that vaccination with VELs based ona survivin-derived CTL epitope induces both CD4+ and CD8+ effectorcytokine-producing T cells and that several variant epitopes lead toincreased T-cell proliferation compared to the FSWT epitope.

Example 5 Immunohistochemical Analysis of VEL-Immunized Mice

VEL-immunized mice were also tested to determine whether they exhibitedmore tumor-infiltrating lymphocytes (TILs) compared to control mice.Tumor tissue sections obtained from mice treated in both prophylacticand therapeutic settings were stained using an anti-CD3 antibody. MoreCD3+ cells were observed in the tumors from mice vaccinated by a singleinoculation with FSVL 40 days before tumor challenge (prophylactictreatment) compared with mice immunized with FSWT, the FB22 controlphage, or with mice transplanted with 4T1 cells without any treatment(data not shown but available upon request). A similar pattern oflymphocytic infiltration was observed in mice vaccinated with theprime/boost regimen (data not shown but available upon request). Also,the presence of TILs was observed in mice vaccinated with correspondingsynthetic peptide immunogens, although the general number of CD3+ Tcells was lower compared with phage-vaccinated mice (data not shown).These data correlated with tumor challenge studies described above,indicating the possible involvement of these TILs in the anti-tumoreffects induced by vaccination with VELs based on a survivin-derived CTLepitope.

Materials and Methods

Variable Epitope Libraries (VELs)

To generate the VELs, molecular biology procedures were carried outusing standard protocols, including the use of restriction enzymes, TaqDNA polymerase, DNA isolation/purification kits, T4 DNA ligase andM13KO7 helper phages. In order to express the survivin-derived wild-typeCTL peptide epitope GWEPDDNPI (SEQ ID NO. 1) and epitope variant-bearingVELs on M13 phage surfaces as fusions with the major phage coat protein(cpVIII), the corresponding DNA fragments were generated by PCR andcloned in a pG8SAET phagemid vector. Briefly, two oligonucleotides(oligos): 5′-gtatattactgtgcgggttgggaaccagatgataatccaatatggggccagggaacc-3′ (SEQ ID NO: 4)and degenerate 5′-gtatattactgtgcgggttgg NNKccaNNKgatNNKccaatatggggccagggaacc-3′ (SEQ ID NO: 5), (N is g, a, t or c and, Kis g or c nucleotide) were used in two separate PCRs with pair ofprimers carrying Nco I and Bam HI restriction sites; 5DAMP:5′-tgatattcgtactcgagccatggtgtatattactgtgcg-3′ (SEQ ID NO: 6) and 3DAMP:5-atgattgacaaagcttggatccctaggttccctggcccca-3 (SEQ ID NO: 7) were used togenerate corresponding DNA fragments for their cloning in phagemidvectors using electroporation. Correct sequences were verified usingstandard automated sequencers.

The resulting recombinant phage clone FSWT expressing the SWT epitopeand the phage library carrying SWT-based VELs, referred as FSVL, wererescued/amplified using M13KO7 helper phages by infection of E. coli TG1cells and purified by double precipitation with polyethylene glycol (20%PEG/2.5 M NaCl). Similarly, 87 phage clones randomly selected from theFSVL library, each expressing different epitope variants, wererescued/amplified from 0.8 mL of bacterial cultures using 96 well 1 mLround bottom blocks. The typical phage yields were 1010 to 1011 colonyforming units (CFU) per milliliter of culture medium. The DNA inserts of27 phage clones from the FSVL library were sequenced and the amino acidsequences of the peptides were deduced, as presented in Table 2 below.

TABLE 2 Sequences of survivin-derived epitope variants Wild-type epitopeSWT G W E P D D N P I Epitope Library G W X^(a) P X D X P I EpitopeVariants  1 — — F — L — A — —  2 — — L — N — Y — —  3 — — R — T — V — — 4 — — F — L — N — —  5 — — I — S — F — —  6 — — Q — T — E — —  7^(b) —— T — K — D — —  8 — — D — L — I — —  9 — — Q — M — S — — 10 — — I — T —A — — 12 — — C — Y — T — — 22 — — N — S — L — — 25 — — V — T — L — — 38— — H — L — N — — 41 — — N — F — G — — 45 — — D — L — Q — — 50 — — A — N— N — — 53 — — V — D — Y — — 58 — — Q — V — T — — 59 — — E — T — H — —65 — — C — Q — L — — 73 — — W — Q — E — — 79 — — F — L — V — — 80 — — V— Y — Y — — 82 — — R — V — P — — 88 — — T — I — R — — Amino acid 14/2612/26 16/26 frequencies ^(a)X-any of the 20 natural amino acids. ^(b)Theclones numbered 7, 10, 12, 22, 25, 38, 41, 45, 50, 53, 58, 59, 65, 73,79, 80, 82 and 88 were used as Ag in T-Cell assays.

The synthetic peptides corresponding to the SWT epitope and the SVLpeptide/epitope library, bearing the complexity of 8×10³ individualmembers, designated as PSWT and PSVL, respectively, as well as a controlpeptide/epitope library PG5D (A[G/F]PXXXXX[L/M]) with theoreticalcomplexity of 3.2×10⁶ individual members were prepared using GenScripttechnology.

Animal Studies

4T1 mouse mammary carcinoma cells (American Type Culture Collection)were maintained for a limited time in vitro by passage in RPMI-1640medium containing 10% FBS and penicillin (100 U/ml), streptomycin (100μg/ml) and fungizone (0.75 μg/ml). Groups of 5-7 female 4 to 6 weeks oldBALB/c mice were used. To generate breast tumors, mice were injectedsubcutaneously (s.c.) with 104 viable 4T1 cells in 50 μL of PBS into theright mammary fat pad. Primary tumors were detected by palpation within1-2 weeks, the mice were observed every 3 days to monitor tumor growth,tumor area was calculated as length×breadth using Vernier calipers andmice were euthanized with CO2 31 days after 4T1 inoculation. For theprevention study, mice were immunized with 5×1012 FSWT, FB22 and FSVLrecombinant M13 phage particles (5×1012 CFU) in 200 μL of PBS byintravenous (i.v.) injection into tail vain, and then mice wereinoculated with 4T1 cells on day 40 post-vaccination. The tumor growthwas monitored as described above. For the established disease study,mice were immunized once with above mentioned phage particles by i.v.injection or with 150 μg of synthetic peptides PSWT, PSVL and PGSD plus150 μg of polynosine-polycytidylic acid (Poly (I:C)) in 100 μl ofphosphate-buffered saline (PBS) by s.c. injection on days 5, 14 and 21of tumor challenge. In separate studies, the mice primed as describedabove received 3 (on days 11, 17 and 23 post-tumor cell injection), 2(on days 17 and 23) or one (on day 26) booster vaccinations with phageor peptide immunogens (5×1011 CFU and 100 μg of synthetic peptides,respectively).

Cell Proliferation Assays

Splenocytes were pooled from 3 animals from each treatment group on day15 after immunization or tumor challenge and tested using standard flowcytometry protocols. Cells were resuspended in RPMI-1640 mediumsupplemented as described above plus 1% sodium pyruvate, 1% nonessentialamino acids and 1% 2-mercaptoethanol, washed twice with PBS andresuspended at 5×107 cells ml—1 in 5 μM CFSE for 10 min at roomtemperature. After washing again two times with 10 mL of PBS+5% FBS at4° C., the cells were stimulated by culturing in a 96-well flat-bottomplate (2.5×105 cells/well) with 1×1010 phage particles/wellcorresponding to particular epitope variant for 72 hrs. at 37° C. in CO2incubator. The gating strategy involved exclusion of doublets and deadcells and, 10,000 lymphocytes (R1) were gated for a CD4+ versus CD8+dot-plot graph to measure CD4+ IFN-γ+, CD8+ IFN-γ+ and proliferationpercentages of CD4+CD8− and CD4−CD8+ cells.

Total cell proliferation and CD4+ and CD8+ T-cell responses wereevaluated by using intracellular staining (ICS) for IFN-γ both ex vivoand in vitro by stimulating fresh lymphocytes for 6 hrs or 72 hrs,respectively. During the last 4 h, 1 μl well Monensin (2 μM) was addedto the culture. The cells were stained with fluorescence-labeledmonoclonal antibodies against CD4 and CD8 for 30 min at roomtemperature, fixed with fixation buffer and, after washing, the cellswere permeabilized with permeabilization wash buffer, then labeled for30 min with anti-IFN-γ antibody in the dark. The cells were analyzed onFACS Calibur Cytometer using CellQuest software; at least 10,000 eventswere collected

Immunohistochemical (IHC) Studies

The tumors were removed at day 31 post-tumor injection. The excisedtumors were fixed in 4% buffered formalin for 12 hrs. at 40° C. Twentymicrometer-thick free-floating sections were processed using standardprotocols. Hydrogen peroxide-quenched and blocked sections wereincubated overnight at 4° C. with anti-CD3 primary antibody (Clone 17A2,dilution 1:500). Sections were washed and incubated with HRP Goatanti-rat IgG antibody (dilution 1:800) for 1 hr at room temperature.After multiple washes, color development was performed using a liquidDAB+substrate chromogen system. Samples were placed onto glass slides,stained with hematoxylin, dehydrated with xylene, and covered withEntellan mounting medium. Samples were viewed on Olympus BX51 microscopeequipped with an Olympus DP71 digital camera.

Statistical Analysis

All results are expressed as the means±s.e.m. Mouse sample group sizeswere at least n=5. All experiments were repeated at least once withcomparable results. Tumor size data were analyzed using repeatedmeasurements analysis with Duncan's “post-hoc” test for multiplecomparisons. Differences were considered significant at P<0.05. Cellproliferation (%) data were analyzed with two tailed t-test. SAS 9.0software was used for statistical analysis. Proportions of epitopevariants were compared using ‘z’ two tailed test; STATISTICA 8.0 wasused in this analysis.

It should also be appreciated that the exemplary embodiment or exemplaryembodiments are only examples, and are not intended to limit the scope,applicability, or configuration of the subject matter hereof in any way.Rather, the foregoing detailed description will provide those skilled inthe art with an enabling disclosure for implementing the exemplaryembodiment or exemplary embodiments. It should be understood thatvarious changes can be made in the function and arrangement of elementswithout departing from the scope of the subject matter hereof as setforth in the appended claims and the legal equivalents thereof.

The embodiments above are intended to be illustrative and not limiting.Additional embodiments are within the claims. Although the presentsubject matter has been described with reference to particularembodiments, workers skilled in the art will recognize that changes maybe made in form and detail without departing from the spirit and scopeof the subject matter hereof.

Various modifications to the subject matter hereof may be apparent toone of skill in the art upon reading this disclosure. For example,persons of ordinary skill in the relevant art will recognize that thevarious features described for the different embodiments of the subjectmatter can be suitably combined, un-combined, and re-combined with otherfeatures, alone, or in different combinations, within the spirit of thesubject matter hereof. Likewise, the various features described aboveshould all be regarded as example embodiments, rather than limitationsto the scope or spirit of the subject matter hereof. Therefore, theabove is not contemplated to limit the scope of the present subjectmatter hereof.

Other features and advantages of the disclosure will be apparent fromthe following detailed description, and from the claims.

All of the COMPOSITIONS and METHODS disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods have been described interms of preferred embodiments, it is apparent to those of skill in theart that variations maybe applied to the COMPOSITIONS and METHODS and inthe steps or in the sequence of steps of the methods described hereinwithout departing from the concept, spirit and scope herein. Morespecifically, certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept as defined bythe appended claims.

What is claimed:
 1. A method of treating cancer in a subject, the methodcomprising: administering a variable epitope library vaccine compositioncomprising one or more synthetic isolated peptides having amino acidsequences corresponding to an epitope of a tumor antigen that isessential for tumor survival and expressed by said tumor at high levels,or nucleic acid encoding said synthetic isolated peptides, said one ormore peptides having from about 7 to about 50 total amino acids, whereinfrom about 1% to about 50% of the total amino acids of the one or morepeptides are variable amino acids, and a pharmaceutically acceptableexcipient; wherein the composition generates an immune response whenadministered to the subject, and wherein said cancer if present in saidsubject, has a mass of less than 10 mm², wherein the tumor antigen issurvivin comprising a CTL epitope, wherein the survivin CTL epitope isthe peptide represented by GWEPDDNPI (SEQ ID NO: 2) having variableamino acids at positions 3, 5 and 7 (GWXPXDXPI (SEQ ID NO: 1)).
 2. Themethod of claim 1, wherein said subject has one or more tumors andwherein said treating the cancer in said subject reduces the size of asaid one or more tumors in said subject.
 3. The method of claim 1,wherein the cancer is metastatic and capable of immune escape.
 4. Themethod of claim 1, wherein the variable amino acids can be any naturallyoccurring amino acids.
 5. The method of claim 1, wherein the totalnumber of different peptides or in the library is about
 87. 6. Themethod of claim 1, wherein the subject has cancer and wherein thecomposition is administered to the subject therapeutically.
 7. Themethod of claim 1, wherein the subject has cancer and wherein thecomposition is administered to the subject therapeutically at a dosefrom about 100 μg to about 1 mg of isolated peptides.
 8. The method ofclaim 1, wherein the subject has cancer and wherein one or more doses ofthe composition are administered to the subject therapeutically atweekly intervals.
 9. The method of claim 1, wherein the epitope of thetumor antigen is the peptide represented by SEQ ID NO: 1 and wherein thetotal number of different peptides in the library is about 20 to about8,000.
 10. The method of claim 1, wherein the variable amino acid atposition 3 is any of Alanine, Cysteine, Aspartate, Glutamate,Phenylalanine, Histidine, Isoleucine, Leucine, Asparagine, Glutamine,Arginine, Threonine, Valine or Tryptophan.
 11. The method of claim 1,wherein the epitope of the tumor antigen is the peptide represented bySEQ ID NO: 1, and wherein the variable amino acid at position 5 is anyof Aspartate, Phenylalanine, Isoleucine, Lysine, Leucine, Methionine,Asparagine, Glutamine, Serine, Threonine, Valine or Tyrosine.
 12. Themethod of claim 1, wherein the epitope of the tumor antigen is thepeptide represented by SEQ ID NO: 1, and wherein the variable amino acidat position 7 is any of Alanine, Aspartate, Glutamate, Phenylalanine,Glycine, Histidine, Isoleucine, Leucine, Asparagine, Proline, Glutamine,Arginine, Serine, Threonine, Valine or Tyrosine.
 13. The method of claim1, wherein the subject has cancer and wherein therapeuticallyadministering to said subject the variable epitope library vaccinecomposition comprising peptides represented by GWXPXDXPI (SEQ ID NO: 1),or nucleic acid encoding said peptides, results in an immune responsecomprising an increased number of CD8+IFN-γ+ cells which recognizevariant survivin-derived CTL epitopes than in the immune responseresulting from administering peptides represented by GWEPDDNPI (SEQ IDNO: 2), or nucleic acid encoding said peptides.